CN115779683B - Virus-removing filtering method - Google Patents
Virus-removing filtering method Download PDFInfo
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- CN115779683B CN115779683B CN202211627037.8A CN202211627037A CN115779683B CN 115779683 B CN115779683 B CN 115779683B CN 202211627037 A CN202211627037 A CN 202211627037A CN 115779683 B CN115779683 B CN 115779683B
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- 238000001914 filtration Methods 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims description 23
- 241000700605 Viruses Species 0.000 claims abstract description 49
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 38
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 29
- 239000012528 membrane Substances 0.000 claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 22
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 claims abstract description 12
- 229960003589 arginine hydrochloride Drugs 0.000 claims abstract description 12
- QZNNVYOVQUKYSC-JEDNCBNOSA-N (2s)-2-amino-3-(1h-imidazol-5-yl)propanoic acid;hydron;chloride Chemical compound Cl.OC(=O)[C@@H](N)CC1=CN=CN1 QZNNVYOVQUKYSC-JEDNCBNOSA-N 0.000 claims abstract description 10
- 238000011068 loading method Methods 0.000 claims abstract description 8
- 238000002386 leaching Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 12
- 239000012527 feed solution Substances 0.000 claims description 5
- 239000000654 additive Substances 0.000 claims description 4
- 230000000996 additive effect Effects 0.000 claims description 3
- 239000000523 sample Substances 0.000 description 39
- 210000004027 cell Anatomy 0.000 description 17
- 230000004907 flux Effects 0.000 description 13
- 238000000855 fermentation Methods 0.000 description 10
- 230000004151 fermentation Effects 0.000 description 10
- 239000000945 filler Substances 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 238000001042 affinity chromatography Methods 0.000 description 6
- 150000001450 anions Chemical class 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 239000012535 impurity Substances 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000002779 inactivation Effects 0.000 description 5
- 239000011148 porous material Substances 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 238000001728 nano-filtration Methods 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 241000699802 Cricetulus griseus Species 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 230000000415 inactivating effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 210000001672 ovary Anatomy 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000004114 suspension culture Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 239000012515 MabSelect SuRe Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 239000012561 harvest cell culture fluid Substances 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000011100 viral filtration Methods 0.000 description 2
- 239000013621 viresolve pro solution Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101000868279 Homo sapiens Leukocyte surface antigen CD47 Proteins 0.000 description 1
- 102100032913 Leukocyte surface antigen CD47 Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
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- 238000004090 dissolution Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
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- 230000007717 exclusion Effects 0.000 description 1
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- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
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- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229920005597 polymer membrane Polymers 0.000 description 1
- 238000011085 pressure filtration Methods 0.000 description 1
- 238000011165 process development Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a virus-removing filtration method, which is a vertical filtration of a flat membrane adopted by low-concentration feed liquid, and comprises rinsing, balancing, loading and leaching, wherein the loading is to load purified protein feed liquid into a virus-removing filtration membrane device for filtration, and the protein feed liquid comprises at least one of arginine hydrochloride and histidine hydrochloride. The scheme of the invention has simple operation, wide applicability and low cost, and can not influence the virus removal rate.
Description
Technical Field
The invention relates to a biopharmaceutical technology, in particular to a virus removal filtering method.
Background
The Chinese pharmacopoeia of 2020 edition and ICH Q5A have clear requirements for virus safety of biological products, products extracted from human and animal tissues or body fluids, animal-derived monoclonal antibodies and recombinant products expressed by eukaryotic cells, and downstream production processes must include a robust virus removal step. The most common virus removal processes include virus inactivation and virus filtration. The virus removal filtration is based on the principle of particle size exclusion, can remove various viruses in a steady way, and can perform a post-process integrity test to ensure the effectiveness of the process, so that the virus removal filtration is widely accepted in the industry.
The virus removal membrane filtration technology is physical interception, so that the influence on the product quality is small, the virus removal membrane filtration technology is complementary with other virus inactivation technologies, is widely applied to the production of biological products for treatment, and realizes virus removal according to the physical interception effect of a membrane with a nano-scale aperture on virus particles, and the virus removal membrane filter usually comprises a high polymer membrane with a nano-scale aperture and mainly depends on the molecular size interception effect, and is also provided with an adsorption mechanism: when the interception function is performed in filtration, substances (such as virus particles) larger than the pore diameter of the membrane are intercepted by the filter membrane; while proteins smaller than the membrane pore size can pass through the membrane pores downstream of the filter membrane. Substances whose adsorption mechanism is partially smaller than the pore size of the membrane may also be adsorbed on the membrane surface or inside the membrane pores by adsorption (e.g., electrostatic adsorption, van der waals forces, hydrogen bonding, etc.).
In process development, the influence of a filter on the treatment rate, flux, yield and quality of feed liquid needs to be examined. The load, filtration rate and recovery rate of virus removal filtration are affected by factors such as the composition of the filtered sample buffer, conductivity, pH, sample purity, sample concentration, sample protein characteristics, etc., which can lead to different degrees of membrane blocking during filtration.
The method for developing the virus-removing filtration process by optimizing the factors is very time-consuming and labor-consuming, has low efficiency, can hardly achieve ideal filtration effect at last, and has high cost, so that the method for improving the filtration flux and the recovery rate remarkably has very important significance for developing the virus-removing filtration process of protein biological products.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Disclosure of Invention
The invention aims to provide a virus removal filtering method, and provides a method for improving the filtering flux of a virus removal filtering membrane, which can effectively solve the problems of lower membrane flux and more protein loss of virus removal filtering, and the provided technical scheme is simple to operate, wide in applicability and low in cost, and can not influence the virus removal rate.
In order to achieve the above objective, the embodiment of the present invention provides a virus removal filtration method, which is a vertical filtration of a flat membrane used for low concentration feed liquid, and includes rinsing, balancing, loading and leaching, wherein the loading is to load purified protein feed liquid on a virus removal filtration membrane device for filtration, and the protein feed liquid includes at least one of arginine hydrochloride and histidine hydrochloride.
In one or more embodiments of the invention, the concentration of protein in the protein feed solution is less than 15mg/ml.
In one or more embodiments of the present invention, arginine hydrochloride is added to the protein feed solution at a concentration of 0.1 to 0.2M.
In one or more embodiments of the present invention, histidine hydrochloride is added to the protein feed solution at a concentration of 0.05-0.1M.
In one or more embodiments of the present invention, 0.1M arginine hydrochloride and 0.05M histidine hydrochloride are added simultaneously to the protein feed solution.
In one or more embodiments of the invention, rinsing is a rinsing of the virus removal filtration membrane device with a rinse solution. The preferred rinse solution is injection water.
In one or more embodiments of the invention, the rinsing is constant pressure filtration: the pressure is controlled at 29psi, the dosage of the rinsing liquid is more than or equal to 100L/m 2 。
In one or more embodiments of the invention, balancing is balancing the virus removal filtration membrane device with a balancing liquid. A preferred equilibration solution is 20mM PB pH 7.0.
In one or more embodiments of the invention, the equilibrium is a constant pressure equilibrium: the pressure is controlled at 29psi, and the dosage of the balancing liquid is more than or equal to 50L/m 2 。
In one or more embodiments of the invention, the rinsing is a top rinsing of the filtration membrane with a rinse solution. A preferred eluent is 20mM PB pH 7.0.
In one or more embodiments of the invention, the constant pressure rinse: the pressure is controlled at 29psi, and the dosage of the leaching solution is more than or equal to 30L/m 2 。
Compared with the prior art, the virus-removing filtering method according to the embodiment of the invention is suitable for the virus-removing filtering process of most recombinant proteins, antibodies or other protein biological products, has simple and convenient operation and is easy for large-scale production; the selection of the dissolution promoting reagent can select histidine hydrochloride and arginine hydrochloride, which are common reagents, and the safety is ensured; the additive does not influence the quality of the protein, and can also protect the stability of the protein; greatly improves the flux of the virus-removing filtration membrane and the yield of samples.
Drawings
FIG. 1 is a graph of antibody A addition of various additives to remove virus filter membrane flux versus load according to an embodiment of the invention;
FIG. 2 is a graph of antibody B addition of a helper reagent to remove viral filter membrane flux versus load according to an embodiment of the present invention;
FIG. 3 is a graph of antibody C virus removal filtration control test flux versus load according to one embodiment of the invention;
FIG. 4 is a graph showing the flux versus load of an antibody C-added arginine hydrochloride virus removal filter with different concentrations according to one embodiment of the invention.
Detailed Description
The following detailed description of embodiments of the invention is, therefore, to be taken in conjunction with the accompanying drawings, and it is to be understood that the scope of the invention is not limited to the specific embodiments.
Example 1
Sample preparation process: plasmid with genes encoding antibody A (double antibody combining CD3 and CD20 targets) is prepared through gene synthesis, the plasmid is introduced into mammalian cells, CHO-K1 (Chinese hamster ovary cells Chinese Hamster Ovary) is selected in the example, a certain number of production cells are obtained through cell expansion, serum-free cell suspension culture is carried out in a 3L fermentation tank, cells with specific genes express the double-specific antibody and are discharged into a culture solution, after 14 days of fermentation, a fermentation solution of the double-specific antibody A is obtained, the double-specific antibody A fermentation solution is subjected to deep filtration to remove some large particulate matters such as cells and cell fragments, and the deep filter adopts D0HC and A1HC of Millipore in series, and the area ratio is 2:1, general inlet flow rate 150LMH, pressure control <14.5psi, and capturing the harvested cell culture fluid by protein a affinity chromatography, wherein the filler is Mabselect SuRe LX, and the affinity chromatography step adopts the recommended condition of the filler. And (3) inactivating the virus of the LowpH after the affinity, adjusting the pH to 5.5 after the inactivation is finished, performing intermediate deep filtration, removing some host cell proteins and host cell DNA, further removing impurities related to the process by adopting anion flow through, taking the Q FF as an anion filler, and collecting the flow through liquid. And next, adopting Capto S Impact for fine purification, removing relevant impurities of the product, mainly removing polymers and fragments, preparing a high-purity sample, and carrying out virus removal filtration.
Virus removal and filtration steps: setting up a virus removal filter device, and adding 20mM PB and 1MArg-HCl with pH 7.0 to Arg-HCl final concentration of 0.2M into the sample to obtain a sample 01; adding 20mM PB and 1M His-HCl to the sample at pH 7.0 to a final concentration of His-HCl of 0.1M to obtain a sample 02; adding 20mM PB, 1M His-HCl pH 7.0 to His-HCl final concentration of 0.05M to the sample as sample 03; 20mM PB, 1M His-HCl pH 7.0 to His-HCl final concentration of 0.08M was added to the sample as sample 04; the virus removal filtration is carried out on the sample of the control experiment without adding auxiliary reagent, the nanofiltration membranes are Viresolve Pro Device of Millipore, the operation steps are shown in table 1, and the filtration effects of the three are shown in figure 1.
TABLE 1 pure Virus filtration step
| Step (a) | Buffer solution | Volume (L/m) 2 ) | Inlet pressure (Psi) |
| Rinsing | Purified water | 100 | ≤29 |
| Balancing | 20mM PB pH 7.0 | 50 | ≤29 |
| Loading sample | N/A | N/A | ≤29 |
| Rinsing | 20mM PB pH 7.0 | 30 | ≤29 |
TABLE 2 summary of virus removal filtration experimental data
TABLE 3 summary of sample quality data after virus removal filtration
As can be seen from FIG. 1, the addition of arginine hydrochloride or histidine hydrochloride to the sample can greatly increase the filtration capacity from 263L/m 2 To greater than 560L/m 2 And the flux decay amplitude is smaller (decay is about 10%) as the experiment proceeds. And after addition, virus removal and filtration are carried out, so that the quality parameters of the sample are not influenced, the method improves the nano-filtration membrane loading capacity, the process keeps higher flux level, and meanwhile, the sample recovery rate is ensured to be more than 98%. The method is simple, does not need a large amount of exploration experiments, is efficient and convenient, and has remarkable effect.
Example 2
Sample preparation process: preparing a plasmid for encoding an antibody B (a double antibody for combining PD1 and CD47 targets) through gene synthesis, introducing the plasmid containing genes for encoding the double-specific antibody B into mammalian cells, selecting CHO-S (Chinese hamster ovary cells Chinese Hamster Ovary) in this example, obtaining a certain number of CHO cells through cell expansion, carrying out serum-free cell suspension culture in a 3L fermentation tank, expressing the double-specific antibody by the cells with specific genes, discharging the double-specific antibody into a culture solution, fermenting for 14 days to obtain a fermentation solution of the double-specific antibody B, removing some large particulate matters such as cells and cell fragments by deep filtration, and connecting D0HC and X0HC of Millipore in series by a deep filter, wherein the area ratio is 2:1, general inlet flow rate 150LMH, pressure control <14.5psi, and capturing the harvested cell culture fluid by protein a affinity chromatography, wherein the filler is Mabselect SuRe LX, and the affinity chromatography step adopts the recommended condition of the filler. And (3) inactivating the virus of the LowpH after the affinity, adjusting the pH to 5.5 after the inactivation is finished, performing intermediate deep filtration, removing some host cell proteins and host cell DNA, further removing impurities related to the process by adopting anion flow through, collecting the flow through liquid, wherein the anion filler is Capto Q. And next, adopting CHT II to carry out fine purification, removing relevant impurities of the product, mainly removing polymers and fragments, preparing a high-purity sample, and carrying out virus removal and filtration.
Adding 20mM PB, 1MArg-HClpH 7.0 and 20mM PB, 1M His-HCl pH 7.0 to the virus-removed filtration sample to a final concentration of 0.1MArg-HCl, 0.05M His-HCl in the system as a sample 11; no auxiliary reagent was added to the control experiment.
TABLE 4 summary of virus removal filtration experimental data
TABLE 5 summary of sample quality data after virus removal filtration
As can be seen from FIG. 2, the addition of arginine hydrochloride and histidine hydrochloride simultaneously to the sample can also greatly increase the filtration capacity from 111L/m 2 To 739L/m 2 And the flux attenuation amplitude is smaller along with the experiment, the nanofiltration membrane loading capacity is improved, and the sample recovery rate is ensured to be more than 99 percent. After the reagent is added, virus removal and filtration are carried out, so that the method has no influence on the quality parameters of the sample, is simple, does not need a large amount of exploration experiments, and is efficient and convenient.
Example 3
Sample preparation process: plasmid encoding antibody C (double antibody binding CD3 and VEGF target) is prepared through gene synthesis, plasmid containing gene encoding bispecific antibody C is introduced into mammal cells, CHO-K1 (Chinese hamster ovary cells Chinese Hamster Ovary) is selected in the example, a certain amount of CHO cells are obtained through cell expansion, serum-free cell suspension culture is carried out in a 3L fermentation tank, cells with specific genes express bispecific antibody and are discharged into a culture solution, fermentation liquid of bispecific antibody C is obtained after 14 days of fermentation, the bispecific antibody C fermentation liquid is subjected to deep filtration to remove some large particulate matters such as cells and cell fragments, D0HC and A1HC of Millipore are adopted in a serial connection mode for a depth filter, and the area ratio is 2:1, general inlet flow rate 150LMH, pressure control <14.5psi, post harvest cell culture broth captured using protein a affinity chromatography using Praesto Jetted a50 as filler, affinity chromatography step using conditions recommended for the filler, eluent 50mM Gly-HCl ph3.5. And (3) inactivating the virus of the Low pH after the affinity, adjusting the pH to 5.5 after the inactivation is finished, performing intermediate deep filtration, removing some host cell proteins and host cell DNA, further removing impurities related to the process by adopting anion flow through, collecting the flow-through liquid, wherein the anion filler is GigaCap Q-650 (M). And next, adopting CHT II to carry out fine purification, removing relevant impurities of the product, mainly removing polymers and fragments, preparing a high-purity sample, and carrying out virus removal and filtration.
Virus removal and filtration steps: 20mM PB, 1M Arg-HCl pH 7.0 to Arg-HCl final concentration of 0.2M was added to the sample as sample 21; 20mM PB, 1MArg-HCl pH 7.0 to Arg-HCl final concentration of 0.1M was added to the sample as sample 22; 20mM PB, 1MArg-HCl pH 7.0 to Arg-HCl final concentration of 0.15M was added to the sample as sample 23; the control samples were subjected to virus removal filtration without the addition of auxiliary reagents, and nanofiltration membranes were each Viresolve Pro Device from Millipore, the procedure being shown in table 1.
TABLE 6 summary of virus removal filtration experimental data
TABLE 7 summary of sample quality data after virus removal filtration
As can be seen from FIGS. 3 and 4, the addition of arginine hydrochloride at different concentrations to the sample greatly increases the filtration capacity from 15L/m 2 To 870L/m 2 And the flux attenuation amplitude is smaller along with the experiment, and the sample recovery rate is ensured to be more than 95 percent. After the reagent is added, virus removal and filtration are carried out, so that the method has no influence on the quality parameters of the sample, is simple, does not need a large amount of exploration experiments, and is efficient and convenient.
The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (2)
1. The virus-removing filtering method is characterized in that the sample loading is to load the purified protein feed liquid into a virus-removing filtering membrane device for filtering, wherein the protein feed liquid also comprises an additive, the additive is at least one of arginine hydrochloride and histidine hydrochloride, the concentration of the arginine hydrochloride added into the protein feed liquid is 0.1-0.2M, the concentration of the histidine hydrochloride added into the protein feed liquid is 0.05-0.1M, the protein concentration in the protein feed liquid is less than 15mg/ml, and the rinsing is constant-pressure filtering: the pressure is controlled at 29psi, the dosage of the rinsing liquid is more than or equal to 100L/m 2 The balance is constant pressure balance: the pressure is controlled at 29psi, and the dosage of the balancing liquid is more than or equal to 50L/m 2 The constant pressure leaching: the pressure is controlled at 29psi, and the dosage of the leaching solution is more than or equal to 30L/m 2 。
2. The method for removing virus according to claim 1, wherein 0.1M arginine hydrochloride and 0.05M histidine hydrochloride are added simultaneously to the protein feed solution.
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| JP2001335509A (en) * | 2000-05-31 | 2001-12-04 | Nihon Pharmaceutical Co Ltd | Method for removing virus from solution containing fibrinogen |
| CN104804078A (en) * | 2015-05-05 | 2015-07-29 | 广东卫伦生物制药有限公司 | Method for filtering viruses in blood coagulation factor VIII of human serum |
| CN105175486A (en) * | 2015-10-20 | 2015-12-23 | 上海洲跃生物科技有限公司 | Preparation method of high-purity human coagulation factor IX |
| CN111116751A (en) * | 2019-10-15 | 2020-05-08 | 北大未名(合肥)生物制药有限公司 | Arginine-based omalizumab anti-aggregation method |
| CN114181300A (en) * | 2021-12-20 | 2022-03-15 | 方坦思(上海)生物医药有限公司 | Preparation method of high-purity monoclonal antibody |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012134987A1 (en) * | 2011-03-25 | 2012-10-04 | Genentech, Inc. | Novel protein purification methods |
| US11052165B2 (en) * | 2015-04-20 | 2021-07-06 | Global Life Sciences Solutions Usa Llc | Method for virus clearance |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001335509A (en) * | 2000-05-31 | 2001-12-04 | Nihon Pharmaceutical Co Ltd | Method for removing virus from solution containing fibrinogen |
| CN104804078A (en) * | 2015-05-05 | 2015-07-29 | 广东卫伦生物制药有限公司 | Method for filtering viruses in blood coagulation factor VIII of human serum |
| CN105175486A (en) * | 2015-10-20 | 2015-12-23 | 上海洲跃生物科技有限公司 | Preparation method of high-purity human coagulation factor IX |
| CN111116751A (en) * | 2019-10-15 | 2020-05-08 | 北大未名(合肥)生物制药有限公司 | Arginine-based omalizumab anti-aggregation method |
| CN114181300A (en) * | 2021-12-20 | 2022-03-15 | 方坦思(上海)生物医药有限公司 | Preparation method of high-purity monoclonal antibody |
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